A vaccine targeting human <scp>IL</scp>‐4 and <scp>IL</scp>‐13 protects against asthma in humanized mice
Authors: Emma Lamanna, Eva Conde, Aurélie Mougel, Jonathan Bonnefoy, Fabien Colaone, Ophélie Godon, Samir Hamdi, Jasper B. J. Kamphuis, Béatrice Drouet, Vincent Serra, Pierre Bruhns, Laurent L. Reber
Journal: Allergy (2023)

Asthma is the most common chronic lung disease, affecting over 300 million people worldwide.1 Among the various potential asthma phenotypes, "type 2-high" asthma is observed in ~50% of asthma patients and is characterized by elevated levels of IgE, type 2 cytokines, such as IL-4 and IL-13, and eosinophilia.1, 2 Although IL-4 and IL-13 share a common receptor subunit (IL-4Rα), the two cytokines have some key nonredundant functions in asthma, with a predominant role for IL-4 in IgE production and for IL-13 in airway remodeling and mucus hypersecretion.3 Dual blockade of IL-4 and IL-13 signaling with the therapeutic anti-IL-4Rα mAb (dupilumab) is efficient at decreasing the rate of severe exacerbations in patients with moderate-to-severe asthma.3 However, given the high cost and the need for frequent reinfusions, the use of this mAb is still limited to a minority of patients. To propose an alternative therapy, we recently reported that using conjugate vaccines (termed kinoids) against both mouse IL-4 and IL-13 can reduce airway hyperresponsiveness (AHR), IgE production, eosinophilia, and mucus production in mouse models of asthma.4, 5 While these results obtained with mouse-specific kinoids represent an important proof-of-concept of the feasibility of such a vaccine approach, it is now necessary to demonstrate the efficacy of kinoids targeting human IL-4 (hIL-4) and hIL-13 in asthma. To do so, we generated hIL-4 kinoids (hIL-4K) and hIL-13K by coupling each of the two cytokines with the carrier protein diphtheria "cross-reactive material 197" (CRM197, a nontoxic mutant of diphtheria toxin).4 We tested the efficacy of a combined vaccination with hIL-4K and hIL-13K in a chronic asthma model induced by a total of 12 intranasal administrations of house dust mite (HDM) extract in mice knocked-in for hIL-4, hIL-13, and hIL-4Rα genes in the place of the respective mouse genes,4 named hIL-4/13/4RαKI mice (Figure S1A). As expected, combined intramuscular vaccination with hIL-4K and hIL-13K emulsified with a squalene-based adjuvant (SWE) induced potent antibody responses against hIL-4 and hIL-13, and also against the carrier protein CRM197 (Figure S1B–D). Importantly, both anti-hIL-4 and anti-hIL-13 antibodies demonstrated strong neutralizing capacity, which was readily observable at day 39 (the earliest time-point we assessed) and lasted at least until day 97, that is until the end of the experiment (Figure 1A,B). HDM-treated hIL-4/13/4RαKI mice immunized with CRM197 emulsified in SWE (as a control) demonstrated elevated eosinophil numbers in bronchoalveolar lavage (BAL) fluid compared with PBS-treated mice. By contrast, HDM-treated mice vaccinated with hIL-4 K/hIL-13 K in SWE had markedly reduced eosinophilia (Figure 1C). By contrast, vaccination with hIL-4 K/hIL-13 K did not affect levels of neutrophils, alveolar macrophages, T cells, and B cells in BAL fluid of HDM-treated mice (Figure S2A–E). Dual vaccination against hIL-4 and hIL-13 also inhibited peribronchiolar inflammation 1.6-fold and reduced mucus production 8.7-fold (Figure 1D–G). We did not observe AHR to inhaled methacholine after repeated HDM exposure in hIL-4/13/4RαKI mice (Figure S2F,G), which is likely due to the genetic background of these mice (129/Sv), in which AHR poorly develops compared with other mouse strains such as BALB/c.6 We, therefore, decided to repeat these asthma experiments by doubling HDM amounts for sensitization and quadrupling them for challenges (termed 'HDMhigh model' Figure S1A). Under these severe conditions, HDM-treated hIL-4/13/4RαKI mice exhibited a marked increase in lung resistance and elastance upon exposure to methacholine, compared with PBS-treated control mice. These two features were nearly abrogated in mice vaccinated with hIL-4 K/hIL-13 K (Figure 1H,I). Mice vaccinated with hIL-4 K/hIL-13 K also exhibited reduced total and HDM-specific IgE levels but had similar levels of HDM-specific IgG compared with HDM-treated controls (Figures 1J–L and S3). Altogether, these results demonstrate that long-term reduction in hIL-4 and hIL-13 activity can be achieved through vaccination with human kinoids and can protect against key features of asthma in humanized mice. Our previous report using a mouse surrogate IL-4/IL-13 kinoids also demonstrated that this dual vaccination strategy is efficient at reducing key asthma features in a therapeutic setting (i.e. when vaccination starts in mice already sensitized to HDM).4 Data on the long-term effects of targeting IL-4, IL-13, or other type 2 cytokines in humans are still limited. However, large clinical studies for the anti-IL-4Rα mAb dupilumab with treatment periods for up to 96 weeks have demonstrated a good safety profile, and in particular, no increase in helminth infection, which argues in favor of the feasibility of long-term targeting of IL-4 and IL-13 with a vaccine.3, 4 It will now be important to further assess the safety and efficacy of this vaccine candidate in nonhuman primates prior to clinical trials. Our results also validate the use of hIL-4/13/4RαKI mice for the assessment of drugs targeting hIL-4, hIL-13, and/or hIL-4Rα in disease models. The authors acknowledge Dr. Livia Brunner and Dr. Nicolas Collin from the Vaccine Formulation Laboratory (Switzerland) for the provision of SWE adjuvant. We are grateful to the staff at the Institut Pasteur Histology Facility (institut pasteur, Paris, France) for technical assistance. Requests regarding hIL-4/13/4RαKI humanized mice should be made to Dr. Beverly Koller (University of North Carolina, USA). E.C. was the recipient of a CIFRE PhD fellowship. P.B. acknowledges support from the European Research Council (ERC)–Seventh Framework Program (ERC-2013-CoG 616,050), and L.L.R. from an ATIP-Avenir grant from the INSERM (Institut National de la Santé et de la Recherche Médicale) and the French Medical Foundation (Equipe FRM 2021). This work was supported by a grant from the French National Research Agency ANR-18-CE18-0023 'AllergyVACS', NEOVACS, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Institut Pasteur. E.C, P.B, V.S., and L.L.R. are inventors on a patent related to this work (WO2019228674 (A1)) licensed to NEOVACS, and/or related to Kinoid technology. E.L., E.C., A.M., J.B., F.C., S.H., B.D., V.S., and L.L.R. are currently or were previously employees of NEOVACS and/or company stock owners. Outside of the submitted work, P.B. received consulting fees from Regeneron Pharmaceuticals, L.L.R. received consulting fees from Novartis and Argenx. The other authors declare no competing interests. Appendix S1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.